This research plan will define how nitric oxide (.NO)-mediated oxidative reactions during inflammatory lung injury lead to the formation of secondary nitrating species that can modify the structure and function of key target molecules. Insight derived from mechanistic studies will be integrated with cell test systems and then extended to a myeloperoxidase- deficient mouse model and pulmonary levels of reactive species specifically modulated. The central hypothesis that underlies the research plan is that pulmonary exposure to endogenous and exogenous nitric oxide, in the face of oxidative stress, leads to the formation of reactive secondary species that alter the function of critical proteins via chemical and peroxidase-catalyzed posttranslational modifications. The identification of these reaction pathways as salutary, benign or injurious is proposed to depend on the extent of formation of modified target molecules, the reversibility of these protein modifications and their impact on cell signaling and metabolic events. There are multiple possible pathways for generating species that nitrate tyrosine residues in proteins, with their relative contributions not well defined; a) the product of superoxide (O2.-) and .NO reaction, peroxynitrite (ONOO-) oxidizes and nitrates proteins, often via CO2 stimulated mechanisms, b) tissue peroxidases react with hydrogen peroxide (H202) to form compound intermediates that oxidize nitrite (NO2-) to a nitrating species and c) the neutrophil myeloperoxidase product hypochlorite (HOCl) oxidizes NO2- to a nitrating species. These two latter pathways may predominate over ONOO- as the proximal mediator of tyrosine nitration, especially when lung NO2- concentrations can climb to approximately 100 micromoles levels during inflammation. Thus, to better understand the molecular pathogenesis of protein modification during inf1ammatory lung injury, the following Specific Aims will be pursued: 1) Define the predominant mechanisms mediating tyrosine nitration in chemical reaction systems and lung cells, 2) Determine the influence of nitric oxide-derived nitrating species on the structure and function of key lung cell proteins and 3) Explore the optimal approaches for limiting the nitration and impaired function of lung cell proteins during inflammatory injury. Upon successful completion of the proposed aims, a) detailed mechanistic information will be available regarding the presence, reactions and regulation of specific oxidative pathways in the lung, b) more will be known about the potential clinical diagnostic utility of oxidative reaction products as molecular "footprints" of specific inflammatory reactions, c) the influence of tyrosine nitration on the function of key pulmonary proteins will be revealed and d) new insight will be gained for prospectively devising mechanism-directed pharmacologic strategies to intervene in pulmonary oxidative inflammatory injury pathways.